Hepatitis E virus vaccine and method

Human Primary Hepatocytes in Culture

A. Isolation of hepatocytes.

Hepatocytes were isolated from human liver obtained from Stanford University Medical Center. The liver was either perfused in situ or excised as a wedge for perfusion in laboratory. The initial perfusion was performed for 10 minutes at 60 ml/min using Ca ++ -, Mg ++ -free Hanks' balanced salt solution supplemented with 10 mM HEPES (pH7.4) and 0.5 mM [ethylene bis(oxyethylenenitrillo]-tetraacetic acid. Perfusion was continued for additional 20 minutes using Williams' medium E (WME) supplemented with 10 Mm HEPES (pH7.4) and 100 U/ml collagenase (type I, Sigma Chemical Co., St. Louis, Mo.).

After perfusion the liver capsule was removed using fine forceps, and hepatocytes were dislodged by gentle shaking in collagenase solution. The hepatocyte suspension was filtered through several layers of gauze and mixed with an equal volume of WMW containing 10% fetal bovine serum (FBS). Hepatocytes were sedimented by centrifugation at 50 ×g for 5 minutes and resuspended in WME containing 5% FBS. Hepatocytes were sedimented and resuspended in the manner for 2 additional times. The final cell preparation was further filtered through several layers of gauze before examining for viability using trypan blue. The cells were plated at a density of 2×10 cells per 60-mm Primaria plates (Falcon) pre-coated with collagen (Collaborative Research).

Cultures were incubated at 37° C. in 5% CO 2 for 3 hours to allow attachment and the medium was changed to a serum-free formulation and every 48 hrs thereafter. The serum-free formulation was a WME-based medium supplemented with growth factors, hormones, 10 mM HEPES (pH7.4), 100 ug/ml gentamycin, as has been described (Lanford).

B. Detection of Liver-Specific Proteins.

Human hepatocyte cultures were maintained in serum-free medium for various period of time and labeled with [ 35 S]-methionine for 24 hrs. The medium was adjusted to contain 1 mM PMSF, 1 mM EDTA, and 1% “NONIDET P-40” detergent (“NP-40”—an octylphenol-ethylene oxide condensate containing an average of 9 moles ethylene oxide per mole of phenol) Antibodies specific for the different plasma proteins were bound to protein A-agarose beads, the beads were washed with PBS, and aliquots of the labeled medium were incubated for 16 hrs at 4° C. with the antibody-bead complexes. The beads were washed 3 times with a buffer containing 1% NP40, and immunoprecipitated proteins were eluted with gel electrophoresis sample buffer containing 2% SDS and 2% 2-mercaptoethanol. Samples were analyzed by gradient SDS-PAGE (4 to 15%) and autoradiography.

In Vitro HEV Infection of Primary human Hepatocytes

A. HEV Infection of human hepatocytes.

The HEV-infected cynomolgus monkey #73 stool pool (fourth passage) was used as an inoculum for infections of primary human hepatocytes. Various amounts of inoculum was diluted in 1 ml of serum-free medium (SFM) and applied to the culture during a 3 hr incubation period. This solution was then supplemented with 2 ml of fresh SFM and the entire mixture was incubated overnight. The next day, cell monolayers were washed with WME (10 mM HEPES, pH7.4) for three times and changed to fresh SFM, which was changed at two day intervals thereafter.

B. Immunofluorescence staining assay.

Primary cynomolgus monkey hepatocytes were isolated and plated in tissue culture plates with collagen-coated coverslips as described. Cells on coverslips were infected with either the HEV-infected cynomolgus monkey #73 stool pool or the NIH normal human serum three days after initial plating. The infections were allowed to proceed for 2 weeks.

Cells on coverslips were fixed in 90% acetone at room temperature for 1 minute. The coverslips were then air-dried. The coverslips were blocked in 1% goat serum in PBS for 1 hour, washed with PBS for three times, and incubated with a mixture of rabbit antisera against HEV recombinant proteins 1L6, 4-2, and 6-1-4 at room temperature for 3 hours. The coverslips were again washed with PBS for 3 times and reacted with fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit IgG(H+L) (Zymed) diluted in PBS-l% goat serum for 30 minutes. After the coverslips were washed with PBS for 3 times and air-dried, they were mounted with FITC glycerol solution and examined under a fluorescent microscope.

C. Reverse transcription/polymerase chain reaction (RT/PCR).

HEV infection of primary cynomolgus macaque hepatocytes was evaluated by RT/PCR assays. The primers for cDNA synthesis and PCR were based on the nucleotide sequences of the full-length HEV cDNA (A. Tam et al.). Primers HEV3.2SF1 (nt 6578-6597) and HEV3.2SF2(nt 6650-6668) are of sense polarity from the ORF2 region of the viral genome and HEV3.2SR1 (nt 7108-7127) and HEV3.2SR2 (nt 7078-7097) are antisense primers within the region.

Following extraction of total cellular RNA from HEV-infected cells using one-step guanidinium procedure or HEV-infected supernatants according to the method of Sherker et al., aliquots of RNA samples were heat-denatured at 95° C. for 5 minutes and subjected to reverse transcription at room temperature for 5 minutes and 42° C. for 60 minutes using 200 units per reaction of MMLV-reverse transcriptase (BRL) in a 20 ul reaction volume containing 20 units of RNasin (Promega), 1×PCR buffer (Perkin-Elmer Cetus), with a concentration of 1 mM each deoxyribonucleotide (Perkin-Elmer Cetus), and 2.5 uM of HEV3.2SR1 primer. The reaction mixture was then heat-treated at 95° C. for 5 minutes to denature the MMLV-reverse transcriptase.

Ten microliters of the cDNA synthesis product was used for PCR in a final volume of 50 ul with 0.5 uM HEV3.2SF1 primer, 1.25 units Taq DNA polymerase (AmpliTaq, Perkin-Elmer Cetus), and 1×PCR buffer, overlayed with 50 ul of mineral oil, and subjected to 40 cycles of PCR in a Perkin-Elmer thermocycler (95° C.×1 minute; 52° C.×2 minutes; 72° C.×30 seconds). Ten microliters of the first-round PCR product then underwent another 40 cycles of nested PCR (95° C.×1 minute; 55° C.×2 minutes; 72° C.×30 seconds) in a total volume of 50 ul containing the internal PCR primers HEV3.2SF2 and HEV3.2SR2.

First- and second-round PCR products were subjected to agarose electrophoresis, ethidium bromide stained and photographed under UV light. The results are shown in FIG. 4, discussed above. Southern transfer was performed and filters were hybridized with [ 32 P-dCTP]-labeled internal probe HEVORF2-7 exclusive of the primers (nt 6782-6997), and autoradiography performed.

Preparation of 406.3-2 and 406.4-2 AntiqGns

A TZKF1 plasmid (ET1.1), ATCC deposit number 67717, was digested with EcoRI to release the 1.33 kb HEV insert which was purified from the linearized plasmid by gel electrophoresis. The purified fragment was suspended in a standard digest buffer (0.5M Tris HCl, pH 7.5; 1 mg/ml BSA; 10 mM MnC12) to a concentration of about 1 mg/ml and digested with DNAse I at room temperature for about 5 minutes. These reaction conditions were determined from a prior calibration study, in which the incubation time required to produce predominantly 100-300 basepair fragments was determined. The material was extracted with phenol/chloroform before ethanol precipitation.

The fragments in the digest mixture were blunt-ended and ligated with EcoRI linkers. The resultant fragments were analyzed by electrophoresis (5-1OV/cm) on 1.2% agarose gel, using PhiX174/HaeIII and lambda/HindIII size markers. The 100-300 bp fraction was eluted onto NA45 strips (Schleicher and Schuell), which were then placed into 1.5 ml microtubes with eluting solution (1 M NaCl, 50 mM arginine, pH 9.0), and incubated at 67° C. for 30-60 minutes. The eluted DNA was phenol/chloroform extracted and then precipitated with two volumes of ethanol. The pellet was resuspended in 20 ml TE (0.01 M Tris HCl, pH 7.5, 0.001 M EDTA).

B. Cloning in an Expression Vector

Lambda gt11 phage vector (Huynh) was obtained from Promega Biotec (Madison, Wis.). This cloning vector has a unique EcoRI cloning site 53 base pairs upstream from the beta-galactosidase translation termination codon. The genomic fragments from above, provided either directly from coding sequences 5) or after amplification of cDNA, were introduced into the EcoRI site by mixing 0.5-1.0 mg EcoRI-cleaved gt11, 0.3-3 ml of the above sized fragments, 0.5 ml lOX ligation buffer (above), 0.5 ml ligase (200 units), and distilled water to 5 ml. The mixture was incubated overnight at 14° C., followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).

The packaged phage were used to infect E. coli strain KM392, obtained from Dr. Kevin Moore, DNAX (Palo Alto, Calif.). Alternatively, E. Coli strain Y1090, available from the American Type Culture Collection (ATCC #37197), could be used. The infected bacteria were plated and the resultant colonies were checked for loss of beta-galactosidase activity-(clear plaques) in the presence of X-gal using a standard X-gal substrate plaque assay method (Maniatis). About 50% of the phage plaques showed loss of beta-galactosidase enzyme activity (recombinants).

C. Screening for HEV Recombinant Proteins

HEV convalescent antiserum was obtained from patients infected during documented HEV outbreaks in Mexico, Borneo, Pakistan, Somalia, and Burma. The sera were immunoreactive with VLPs in stool specimens from each of several other patients with ETNANB hepatitis.

A lawn of E. coli KM392 cells infected with about 104 pfu of the phage stock from above was prepared on a 150 mm plate and incubated, inverted, for 5-8 hours at 37° C. The lawn was overlaid with a nitrocellulose sheet, causing transfer of expressed HEV recombinant protein from the plaques to the paper. The plate and filter were indexed for matching corresponding plate and filter positions.

The filter was washed twice in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% “TWEEN 20” (a polyoxyethylenesorbitan monolaurate with a fatty acid composition of approximately 55% lauric acid, with a balance composed primarily of myristic, palmitic and steric acids), blocked with AIB (TBST buffer with 1% gelatin), washed again in TBST, and incubated overnight after addition of antiserum (diluted to 1:50 in AIB, 12-15 ml/plate). The sheet was washed twice in TBST and then contacted with enzyme-labeled anti-human antibody to attach the labeled antibody at filter sites containing antigen recognized by the antiserum. After a final washing, the filter was developed in a substrate medium containing 33 ml NBT (50 mg/ml stock solution maintained at 4° C.) mixed with 16 ml BCIP (50 mg/ml stock solution maintained at 4° C.) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12). Purple color appeared at points of antigen production, as recognized by the antiserum.

D. Screening Plating

The areas of antigen production determined in the previous step were replated at about 100-200 pfu on an 82 mm plate. The above steps, beginning with a 5-8 hour incubation, through NBT-BCIP development, were repeated in order to plaque purify phage secreting an antigen capable of reacting with the HEV antibody. The identified plaques were picked and eluted in phage buffer (Maniatis, p. 443).

Two subclones which were selected are the 406.3-2 and 406.4-2 clones whose sequences are set forth above. These sequences were isolated from an amplified cDNA library derived from a Mexican stool. Using the techniques described in this section, polypeptides expressed by these clones have been tested for immunoreactivity against a number of different human HEV-positive sera obtained from sources around the world. As shown in Table 4 below, 8 sera immunoreactive with the polypeptide expressed by the 406.4-2, and 6 sera immunoreacted with polypeptide expressed by the 406.3-2 clone.

For comparison, the Table also shows reactivity of the various human sera with the non structural peptide Y2. Only one of the sera reacted with the polypeptide expressed by this clone. No immunoreactivity was seen for normal expression products of the gt11 vector.

E. Producing the 406.3-2 Antigen

The 406.3-2 gt11 plasmid from above was digested with EcoRI and the released HEV fragment was amplified by PCR in the presence of linkers which added an NcoI site at the 5′ fragment end, and a BamHI site at the 3′ fragment end. The amplified material was digested with NcoI and BamHI and inserted into the NcoI/BamHI site of the glutathione S-transferase vector pGEX expression vector, according to the manufacturer's instructions.

The pGEX plasmid was used to transform E.coli host cells, and cells which were successfully transformed with the pGEX vector were identified by immunofluorescence, using anti-HEV human antisera.

F. Producing the 406.4-2 Antigen

The 406.4-2 gt11 plasmid from above was digested with EcoRI and the released HEV fragment was amplified by PCR, and the amplified fragment was inserted into the NcoI/BamHI site of the PGEX expression vector, as above. Peptide expression of the 406.4-2 peptide was similar to that described for the 406.3.2 fusion peptide.

G. Preparing Antibodies

The 406.3-2(M) and 406.4-2(M) fusion proteins, prepared as above, were used to immunize rabbits to generate HEV-specific antisera, according to standard procedures.

Neutralizing Activity of Anti-3.2(M) Antibody

A. In vitro Infection

To prove that primary human hepatocytes were permissive for HEV infection and replication, cells were exposed to either normal human serum (NIH normal human serum pool) or HEV-infected cynomolgus macaque stool preparation (cyno#73). Fourteen days postinfection, total cellular RNAs were prepared for reverse-transcription (RT)/polymerase chain reaction (PCR) assays to evaluate the infectability of primary human hepatocytes with HEV. The results indicated that primary human hepatocytes were capable of supporting HEV propagation (FIG. 4 ).

Although quantitative PCR was not applied, total cellular RNA isolated from HEV-infected primary human hepatocytes would indicate a high level of virus replication as suggested by the extent of hybridization with the α- 32 P-dCTP labeled HEV-specific probe (lane 5). There was no evidence of HEV in total cellular RNA isolated from primary human hepatocytes treated with normal human serum pool (lane 4). As negative controls for RT/PCR assays, no carry-over or cross-contamination was detected (lanes 1, 2, and 3). The original HEV-infected cynomolgus macaque stool (cyno#73) was served as a positive control in the RT/PCR assays (lane 6).

B. Neutralizing Activity of Antibody

To examine the neutralizing activities of anti-3-2(M), -4-2-(M), each rabbit antiserum was used at a final dilution of 1:20 with the viral inoculum for HEV infection of primary human hepatocytes. The diluted antibody and viral inoculum were incubated together prior to infection of the cultured cells. Rabbit anti-3-2(M) exhibited a high level of neutralizing activity against HEV infection (FIG. 5, lane 2 versus lane 1). Very little neutralizing activity was observed in rabbit anti-4-2(M) (lane 4 versus lane 3).

This result suggests that the HEV 3-2(M) but not HEV 4-2(M) or 4-2(B) recombinant protein encoded a neutralizing epitope capable of eliciting protective antibody or antibodies against HEV infection. The fact that the Mexico clone 3-2(M) and the Burma clone 3-2(B) share 90.5% homology at the amino acid level (79.8% at the nucleotide level) suggested that antibody(ies) raised against 3-2(M) should cross-neutralize or cross-protect HEV of Mexico or Burma strain from infecting permissive cells.

Neutralizing Activity of Anti-3-2(B) and Anti-4-2(B)

HEV type-common epitopes 3-2 and 4-2 of Burma (B) or Mexico (M) strains were previously identified by screening high titer lambda library for HEV-specific antigen-producing clones using convalescent human serum F387-C. The lambda gt11 clones, 406.3-2 and 406.4-2, were characterized and subcloned to express as betagalactosidase fusion proteins. These fusion proteins were subsequently used to immunize rabbits to generate HEV-specific antisera.

To examine the neutralizing activities of anti-3-2(B) and anti-4-2(B), preimmune rabbit serum or rabbit anti-3-2(B) or anti-4-2(B) antiserum was used at a final dilution of 1:20 with the viral inoculum of Burma strain for HEV invection of primary cynomolgus macaque hepatocytes. Both rabbit anti-3-2(B) (FIG. 10, lane 2) and anti-4-2(B) (FIG. 10, lane 4) but not rabbit preimmune serum (FIG. 10, lane 1 or lane 3) exhibited extraordinary levels of neutralizing activity against HEV infection as indicated by RT/PCR analysis (FIG. 10 panels A and B). This result indicated that both HEV 3-2(B)(Sequence ID No. 13) and HEV 4-2(B)(Sequence ID No. 22) recombinant proteins encode a neutralizing epitope capable of eliciting protective antibody or antibodies against HEV infection. The neutralizing activity of anti-4-2(B) was previously not shown. Therefore, in a cynomologus macaque hepatocyte system it has now been shown that rabbit anti-4-2(B) antibody will neutralize HEV. Thus, the HEV protein designated by sequence ID No 22 is suitable as an immunogen against HEV.

Vaccine Protection Against HEV

A. Preparation of trpE-C2 peptide

The pBET1 plasmid containing a 2.3 kb insert, corresponding to the 1.8 kb 3′ end portion of HEV has been described (Tam). The plasmid was digested with EcoRI, releasing two HEV fragments having sizes of about 600 bp and 1400 bp of which 1210 bp contain coding sequence. The larger fragment was purified by electrophoresis and inserted into the EcoRI site of the pATH10 trpE fusion vector, obtained from T. J. Koerner et al. (Department of Microbiology, UCLA). The recombinant vector was used to transform E. coli DH5αF'host.

The recombinant typE-C2 fusion protein from pATH C2 was isolated by a modification of the procedure of Dieckmann et al. The bacterium containing the pATH C2 plasmid was grown overnight in growth media containing tryptophane. Two ml of the overnight culture was inoculated into 100 ml of fresh growth media and grown at 37° C. for an additional four hours. The bacterial broth was added to one liter of fresh growth media without tryptophane and allowed to grow at 30° C. for 1.5 hours. Ten ml indoleacrylic acid (1 mg/ml) was added and growth was continued for an additional 5 to 10 hours at 30° C. The bacterial cells were collected by centrifugation. The cell pellet was resuspended in a hypotonic solution containing lysozyme to degrade the bacterial cell wall. Sodium chloride and the detergent “NP-40” were added to the suspension to cause hypertonic lysis of the cells. The lysed cell solution was sonicated. The solution was centrifuged. The resulting protein pellet was resuspended in about 5 ml of 10 mM Tris pH 7.5 using a dounce homogenizer. Approximately 75% of the protein in solution was composed of the trpE-C2 protein.

B. Preparation of Vaccine

Converted alum adjuvant was prepared according to standard methods. Briefly, a 10% alum suspension was titrated to pH 6.6 with 1N NaOH, then stirred overnight at 4° C. The suspension is clarified by low-speed centrifugation, and the supernatant decanted. A small amount of 0.9% NaCl+1:20,000 formalin was added to each pellet, and suspended by vortexing. To prepare an antigen vaccine composition, trpE-C2 fusion protein from above is added in a 0.9% NaCl solution to a desired final antigen concentration.

A non-adjuvanted insoluble trpE-C2 peptide was prepared as above in section A.

C. Vaccination

Six cynomolgus monkeys, designated 8901, 8902, 8903, 8910, 9902, and 9904, were used in the vaccination study. Four of the monkeys, 8901, and 8902 8903, and 8910 were immunized by intravenous injection with 1.0 ml of the alum adjuvanted trpE-C2 composition (containing about 50 μg of C2 peptide). The other two animals received adjuvant only. One month later the six animals were given a second vaccination, identical to the first. 4 weeks after the second vaccination, saer from the animals was tested for anti-HEV antibodies by Western blotting, using a fusionless C2 protein. At this stage, animals 8901 and 8902 each received a third vaccination with the non-adjuvanted, insoluble trpE-C2 composition (a total IV dose of about 80 μg trpE-C2 peptide each), and both animals showed anti-HEV by Western blotting 4 weeks later.

Animals 8901, 8903, and 9002 were each challenged IV with 1 ml each of a 10% third passage cyno stool (Burma strain) previously shown to be highly infectious. Animals 8902, 8910, and 9004 were each challenged IV with 1 ml of a proven infectious human stool isolate, Mexican #14, known to cause severe disease in cynos and moderate disease in chimpanzees. The results are shown in FIGS. 2A, 2 B, and 3 A, and 3 B, discussed above.

While the invention has been described with reference to particular embodiments, methods, construction and use, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

22

2094 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

BURMA SEQUENCE, FIGURE 7

1

TGGAATGAAT AACATGTCTT TTGCTGCGCC CATGGGTTCG CGACCATGCG CCCTCGGCCT 60

ATTTTGTTGC TGCTCCTCAT GTTTTTGCCT ATGCTGCCCG CGCCACCGCC CGGTCAGCCG 120

TCTGGCCGCC GTCGTGGGCG GCGCAGCGGC GGTTCCGGCG GTGGTTTCTG GGGTGACCGG 180

GTTGATTCTC AGCCCTTCGC AATCCCCTAT ATTCATCCAA CCAACCCCTT CGCCCCCGAT 240

GTCACCGCTG CGGCCGGGGC TGGACCTCGT GTTCGCCAAC CCGCCCGACC ACTCGGCTCC 300

GCTTGGCGTG ACCAGGCCCA GCGCCCCGCC GTTGCCTCAC GTCGTAGACC TACCACAGCT 360

GGGGCCGCGC CGCTAACCGC GGTCGCTCCG GCCCATGACA CCCCGCCAGT GCCTGATGTC 420

GACTCCCGCG GCGCCATCTT GCGCCGGCAG TATAACCTAT CAACATCTCC CCTTACCTCT 480

TCCGTGGCCA CCGGCACTAA CCTGGTTCTT TATGCCGCCC CTCTTAGTCC GCTTTTACCC 540

CTTCAGGACG GCACCAATAC CCATATAATG GCCACGGAAG CTTCTAATTA TGCCCAGTAC 600

CGGGTTGCCC GTGCCACAAT CCGTTACCGC CCGCTGGTCC CCAATGCTGT CGGCGGTTAC 660

GCCATCTCCA TCTCATTCTG GCCACAGACC ACCACCACCC CGACGTCCGT TGATATGAAT 720

TCAATAACCT CGACGGATGT TCGTATTTTA GTCCAGCCCG GCATAGCCTC TGAGCTTGTG 780

ATCCCAAGTG AGCGCCTACA CTATCGTAAC CAAGGCTGGC GCTCCGTCGA GACCTCTGGG 840

GTGGCTGAGG AGGAGGCTAC CTCTGGTCTT GTTATGCTTT GCATACATGG CTCACTCGTA 900

AATTCCTATA CTAATACACC CTATACCGGT GCCCTCGGGC TGTTGGACTT TGCCCTTGAG 960

CTTGAGTTTC GCAACCTTAC CCCCGGTAAC ACCAATACGC GGGTCTCCCG TTATTCCAGC 1020

ACTGCTCGCC ACCGCCTTCG TCGCGGTGCG GACGGGACTG CCGAGCTCAC CACCACGGCT 1080

GCTACCCGCT TTATGAAGGA CCTCTATTTT ACTAGTACTA ATGGTGTCGG TGAGATCGGC 1140

CGCGGGATAG CCCTCACCCT GTTCAACCTT GCTGACACTC TGCTTGGCGG CCTGCCGACA 1200

GAATTGATTT CGTCGGCTGG TGGCCAGCTG TTCTACTCCC GTCCCGTTGT CTCAGCCAAT 1260

GGCGAGCCGA CTGTTAAGTT GTATACATCT GTAGAGAATG CTCAGCAGGA TAAGGGTATT 1320

GCAATCCCGC ATGACATTGA CCTCGGAGAA TCTCGTGTGG TTATTCAGGA TTATGATAAC 1380

CAACATGAAC AAGATCGGCC GACGCCTTCT CCAGCCCCAT CGCGCCCTTT CTCTGTCCTT 1440

CGAGCTAATG ATGTGCTTTG GCTCTCTCTC ACCGCTGCCG AGTATGACCA GTCCACTTAT 1500

GGCTCTTCGA CTGGCCCAGT TTATGTTTCT GACTCTGTGA CCTTGGTTAA TGTTGCGACC 1560

GGCGCGCAGG CCGTTGCCCG GTCGCTCGAT TGGACCAAGG TCACACTTGA CGGTCGCCCC 1620

CTCTCCACCA TCCAGCAGTA CTCGAAGACC TTCTTTGTCC TGCCGCTCCG CGGTAAGCTC 1680

TCTTTCTGGG AGGCAGGCAC AACTAAAGCC GGGTACCCTT ATAATTATAA CACCACTGCT 1740

AGCGACCAAC TGCTTGTCGA GAATGCCGCC GGGCACCGGG TCGCTATTTC CACTTACACC 1800

ACTAGCCTGG GTGCTGGTCC CGTCTCCATT TCTGCGGTTG CCGTTTTAGC CCCCCACTCT 1860

GCGCTAGCAT TGCTTGAGGA TACCTTGGAC TACCCTGCCC GCGCCCATAC TTTTGATGAT 1920

TTCTGCCCAG AGTGCCGCCC CCTTGGCCTT CAGGGCTGCG CTTTCCAGTC TACTGTCGCT 1980

GAGCTTCAGC GCCTTAAGAT GAAGGTGGGT AAAACTCGGG AGTTGTAGTT TATTTGCTTG 2040

TGCCCCCCTT CTTTCTGTTG CTTATTTCTC ATTTCTGCGT TCCGCGCTCC CTGA 2094

2100 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

MEXICO, FIGURE 7

2

CTGAATGAAT AACATGTGGT TTGCTGCGCC CATGGGTTCG CCACCATGCG CCCTAGGCCT 60

CTTTTGCTGT TGTTCCTCTT GTTTCTGCCT ATGTTGCCCG CGCCACCGAC CGGTCAGCCG 120

TCTGGCCGCC GTCGTGGGCG GCGCAGCGGC GGTACCGGCG GTGGTTTCTG GGGTGACCGG 180

GTTGATTCTC AGCCCTTCGC AATCCCCTAT ATTCATCCAA CCAACCCCTT TGCCCCAGAC 240

GTTGCCGCTG CGTCCGGGTC TGGACCTCGC CTTCGCCAAC CAGCCCGGCC ACTTGGCTCC 300

ACTTGGCGAG ATCAGGCCCA GCGCCCCTCC GCTGCCTCCC GTCGCCGACC TGCCACAGCC 360

GGGGCTGCGG CGCTGACGGC TGTGGCGCCT GCCCATGACA CCTCACCCGT CCCGGACGTT 420

GATTCTCGCG GTGCAATTCT ACGCCGCCAG TATAATTTGT CTACTTCACC CCTGACATCC 480

TCTGTGGCCT CTGGCACTAA TTTAGTCCTG TATGCAGCCC CCCTTAATCC GCCTCTGCCG 540

CTGCAGGACG GTACTAATAC TCACATTATG GCCACAGAGG CCTCCAATTA TGCACAGTAC 600

CGGGTTGCCC GCGCTACTAT CCGTTACCGG CCCCTAGTGC CTAATGCAGT TGGAGGCTAT 660

GCTATATCCA TTTCTTTCTG GCCTCAAACA ACCACAACCC CTACATCTGT TGACATGAAT 720

TCCATTACTT CCACTGATGT CAGGATTCTT GTTCAACCTG GCATAGCATC TGAATTGGTC 780

ATCCCAAGCG AGCGCCTTCA CTACCGCAAT CAAGGTTGGC GCTCGGTTGA GACATCTGGT 840

GTTGCTGAGG AGGAAGCCAC CTCCGGTCTT GTCATGTTAT GCATACATGG CTCTCCAGTT 900

AACTCCTATA CCAATACCCC TTATACCGGT GCCCTTGGCT TACTGGACTT TGCCTTAGAG 960

CTTGAGTTTC GCAATCTCAC CACCTGTAAC ACCAATACAC GTGTGTCCCG TTACTCCAGC 1020

ACTGCTCGTC ACTCCGCCCG AGGGGCCGAC GGGACTGCGG AGCTGACCAC AACTGCAGCC 1080

ACCAGGTTCA TGAAAGATCT CCACTTTACC GGCCTTAATG GGGTAGGTGA AGTCGGCCGC 1140

GGGATAGCTC TAACATTACT TAACCTTGCT GACACGCTCC TCGGCGGGCT CCCGACAGAA 1200

TTAATTTCGT CGGCTGGCGG GCAACTGTTT TATTCCCGCC CGGTTGTCTC AGCCAATGGC 1260

GAGCCAACCG TGAAGCTCTA TACATCAGTG GAGAATGCTC AGCAGGATAA GGGTGTTGCT 1320

ATCCCCCACG ATATCGATCT TGGTGATTCG CGTGTGGTCA TTCAGGATTA TGACAACCAG 1380

CATGAGCAGG ATCGGCCCAC CCCGTCGCCT GCGCCATCTC GGCCTTTTTC TGTTCTCCGA 1440

GCAAATGATG TACTTTGGCT GTCCCTCACT GCAGCCGAGT ATGACCAGTC CACTTACGGG 1500

TCGTCAACTG GCCCGGTTTA TATCTCGGAC AGCGTGACTT TGGTGAATGT TGCGACTGGC 1560

GCGCAGGCCG TAGCCCGATC GCTTGACTGG TCCAAAGTCA CCCTCGACGG GCGGCCCCTC 1620

CCGACTGTTG AGCAATATTC CAAGACATTC TTTGTGCTCC CCCTTCGTGG CAAGCTCTCC 1680

TTTTGGGAGG CCGGCACAAC AAAAGCAGGT TATCCTTATA ATTATAATAC TACTGCTAGT 1740

GACCAGATTC TGATTGAAAA TGCTGCCGGC CATCGGGTCG CCATTTCAAC CTATACCACC 1800

AGGCTTGGGG CCGGTCCGGT CGCCATTTCT GCGGCCGCGG TTTTGGCTCC ACGCTCCGCC 1860

CTGGCTCTGC TGGAGGATAC TTTTGATTAT CCGGGGCGGG CGCACACATT TGATGACTTC 1920

TGCCCTGAAT GCCGCGCTTT AGGCCTCCAG GGTTGTGCTT TCCAGTCAAC TGTCGCTGAG 1980

CTCCAGCGCC TTAAAGTTAA GGTGGGTAAA ACTCGGGAGT TGTAGTTTAT TTGGCTGTGC 2040

CCACCTACTT ATATCTGCTG ATTTCCTTTA TTTCCTTTTT CTCGGTCCCG CGCTCCCTGA 2100

2049 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

ORF 2, BURMA, FIGURE 7

3

ATGCGCCCTC GGCCTATTTT GTTGCTGCTC CTCATGTTTT TGCCTATGCT GCCCGCGCCA 60

CCGCCCGGTC AGCCGTCTGG CCGCCGTCGT GGGCGGCGCA GCGGCGGTTC CGGCGGTGGT 120

TTCTGGGGTG ACCGGGTTGA TTCTCAGCCC TTCGCAATCC CCTATATTCA TCCAACCAAC 180

CCCTTCGCCC CCGATGTCAC CGCTGCGGCC GGGGCTGGAC CTCGTGTTCG CCAACCCGCC 240

CGACCACTCG GCTCCGCTTG GCGTGACCAG GCCCAGCGCC CCGCCGTTGC CTCACGTCGT 300

AGACCTACCA CAGCTGGGGC CGCGCCGCTA ACCGCGGTCG CTCCGGCCCA TGACACCCCG 360

CCAGTGCCTG ATGTCGACTC CCGCGGCGCC ATCTTGCGCC GGCAGTATAA CCTATCAACA 420

TCTCCCCTTA CCTCTTCCGT GGCCACCGGC ACTAACCTGG TTCTTTATGC CGCCCCTCTT 480

AGTCCGCTTT TACCCCTTCA GGACGGCACC AATACCCATA TAATGGCCAC GGAAGCTTCT 540

AATTATGCCC AGTACCGGGT TGCCCGTGCC ACAATCCGTT ACCGCCCGCT GGTCCCCAAT 600

GCTGTCGGCG GTTACGCCAT CTCCATCTCA TTCTGGCCAC AGACCACCAC CACCCCGACG 660

TCCGTTGATA TGAATTCAAT AACCTCGACG GATGTTCGTA TTTTAGTCCA GCCCGGCATA 720

GCCTCTGAGC TTGTGATCCC AAGTGAGCGC CTACACTATC GTAACCAAGG CTGGCGCTCC 780

GTCGAGACCT CTGGGGTGGC TGAGGAGGAG GCTACCTCTG GTCTTGTTAT GCTTTGCATA 840

CATGGCTCAC TCGTAAATTC CTATACTAAT ACACCCTATA CCGGTGCCCT CGGGCTGTTG 900

GACTTTGCCC TTGAGCTTGA GTTTCGCAAC CTTACCCCCG GTAACACCAA TACGCGGGTC 960

TCCCGTTATT CCAGCACTGC TCGCCACCGC CTTCGTCGCG GTGCGGACGG GACTGCCGAG 1020

CTCACCACCA CGGCTGCTAC CCGCTTTATG AAGGACCTCT ATTTTACTAG TACTAATGGT 1080

GTCGGTGAGA TCGGCCGCGG GATAGCCCTC ACCCTGTTCA ACCTTGCTGA CACTCTGCTT 1140

GGCGGCCTGC CGACAGAATT GATTTCGTCG GCTGGTGGCC AGCTGTTCTA CTCCCGTCCC 1200

GTTGTCTCAG CCAATGGCGA GCCGACTGTT AAGTTGTATA CATCTGTAGA GAATGCTCAG 1260

CAGGATAAGG GTATTGCAAT CCCGCATGAC ATTGACCTCG GAGAATCTCG TGTGGTTATT 1320

CAGGATTATG ATAACCAACA TGAACAAGAT CGGCCGACGC CTTCTCCAGC CCCATCGCGC 1380

CCTTTCTCTG TCCTTCGAGC TAATGATGTG CTTTGGCTCT CTCTCACCGC TGCCGAGTAT 1440

GACCAGTCCA CTTATGGCTC TTCGACTGGC CCAGTTTATG TTTCTGACTC TGTGACCTTG 1500

GTTAATGTTG CGACCGGCGC GCAGGCCGTT GCCCGGTCGC TCGATTGGAC CAAGGTCACA 1560

CTTGACGGTC GCCCCCTCTC CACCATCCAG CAGTACTCGA AGACCTTCTT TGTCCTGCCG 1620

CTCCGCGGTA AGCTCTCTTT CTGGGAGGCA GGCACAACTA AAGCCGGGTA CCCTTATAAT 1680

TATAACACCA CTGCTAGCGA CCAACTGCTT GTCGAGAATG CCGCCGGGCA CCGGGTCGCT 1740

ATTTCCACTT ACACCACTAG CCTGGGTGCT GGTCCCGTCT CCATTTCTGC GGTTGCCGTT 1800

TTAGCCCCCC ACTCTGCGCT AGCATTGCTT GAGGATACCT TGGACTACCC TGCCCGCGCC 1860

CATACTTTTG ATGATTTCTG CCCAGAGTGC CGCCCCCTTG GCCTTCAGGG CTGCGCTTTC 1920

CAGTCTACTG TCGCTGAGCT TCAGCGCCTT AAGATGAAGG TGGGTAAAAC TCGGGAGTTG 1980

TAGTTTATTT GCTTGTGCCC CCCTTCTTTC TGTTGCTTAT TTCTCATTTC TGCGTTCCGC 2040

GCTCCCTGA 2049

2055 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

ORF 2, MEXICO, FIGURE 7

4

ATGCGCCCTA GGCCTCTTTT GCTGTTGTTC CTCTTGTTTC TGCCTATGTT GCCCGCGCCA 60

CCGACCGGTC AGCCGTCTGG CCGCCGTCGT GGGCGGCGCA GCGGCGGTAC CGGCGGTGGT 120

TTCTGGGGTG ACCGGGTTGA TTCTCAGCCC TTCGCAATCC CCTATATTCA TCCAACCAAC 180

CCCTTTGCCC CAGACGTTGC CGCTGCGTCC GGGTCTGGAC CTCGCCTTCG CCAACCAGCC 240

CGGCCACTTG GCTCCACTTG GCGAGATCAG GCCCAGCGCC CCTCCGCTGC CTCCCGTCGC 300

CGACCTGCCA CAGCCGGGGC TGCGGCGCTG ACGGCTGTGG CGCCTGCCCA TGACACCTCA 360

CCCGTCCCGG ACGTTGATTC TCGCGGTGCA ATTCTACGCC GCCAGTATAA TTTGTCTACT 420

TCACCCCTGA CATCCTCTGT GGCCTCTGGC ACTAATTTAG TCCTGTATGC AGCCCCCCTT 480

AATCCGCCTC TGCCGCTGCA GGACGGTACT AATACTCACA TTATGGCCAC AGAGGCCTCC 540

AATTATGCAC AGTACCGGGT TGCCCGCGCT ACTATCCGTT ACCGGCCCCT AGTGCCTAAT 600

GCAGTTGGAG GCTATGCTAT ATCCATTTCT TTCTGGCCTC AAACAACCAC AACCCCTACA 660

TCTGTTGACA TGAATTCCAT TACTTCCACT GATGTCAGGA TTCTTGTTCA ACCTGGCATA 720

GCATCTGAAT TGGTCATCCC AAGCGAGCGC CTTCACTACC GCAATCAAGG TTGGCGCTCG 780

GTTGAGACAT CTGGTGTTGC TGAGGAGGAA GCCACCTCCG GTCTTGTCAT GTTATGCATA 840

CATGGCTCTC CAGTTAACTC CTATACCAAT ACCCCTTATA CCGGTGCCCT TGGCTTACTG 900

GACTTTGCCT TAGAGCTTGA GTTTCGCAAT CTCACCACCT GTAACACCAA TACACGTGTG 960

TCCCGTTACT CCAGCACTGC TCGTCACTCC GCCCGAGGGG CCGACGGGAC TGCGGAGCTG 1020

ACCACAACTG CAGCCACCAG GTTCATGAAA GATCTCCACT TTACCGGCCT TAATGGGGTA 1080

GGTGAAGTCG GCCGCGGGAT AGCTCTAACA TTACTTAACC TTGCTGACAC GCTCCTCGGC 1140

GGGCTCCCGA CAGAATTAAT TTCGTCGGCT GGCGGGCAAC TGTTTTATTC CCGCCCGGTT 1200

GTCTCAGCCA ATGGCGAGCC AACCGTGAAG CTCTATACAT CAGTGGAGAA TGCTCAGCAG 1260

GATAAGGGTG TTGCTATCCC CCACGATATC GATCTTGGTG ATTCGCGTGT GGTCATTCAG 1320

GATTATGACA ACCAGCATGA GCAGGATCGG CCCACCCCGT CGCCTGCGCC ATCTCGGCCT 1380

TTTTCTGTTC TCCGAGCAAA TGATGTACTT TGGCTGTCCC TCACTGCAGC CGAGTATGAC 1440

CAGTCCACTT ACGGGTCGTC AACTGGCCCG GTTTATATCT CGGACAGCGT GACTTTGGTG 1500

AATGTTGCGA CTGGCGCGCA GGCCGTAGCC CGATCGCTTG ACTGGTCCAA AGTCACCCTC 1560

GACGGGCGGC CCCTCCCGAC TGTTGAGCAA TATTCCAAGA CATTCTTTGT GCTCCCCCTT 1620

CGTGGCAAGC TCTCCTTTTG GGAGGCCGGC ACAACAAAAG CAGGTTATCC TTATAATTAT 1680

AATACTACTG CTAGTGACCA GATTCTGATT GAAAATGCTG CCGGCCATCG GGTCGCCATT 1740

TCAACCTATA CCACCAGGCT TGGGGCCGGT CCGGTCGCCA TTTCTGCGGC CGCGGTTTTG 1800

GCTCCACGCT CCGCCCTGGC TCTGCTGGAG GATACTTTTG ATTATCCGGG GCGGGCGCAC 1860

ACATTTGATG ACTTCTGCCC TGAATGCCGC GCTTTAGGCC TCCAGGGTTG TGCTTTCCAG 1920

TCAACTGTCG CTGAGCTCCA GCGCCTTAAA GTTAAGGTGG GTAAAACTCG GGAGTTGTAG 1980

TTTATTTGGC TGTGCCCACC TACTTATATC TGCTGATTTC CTTTATTTCC TTTTTCTCGG 2040

TCCCGCGCTC CCTGA 2055

147 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

406.3-2, BURMA, FIGURE 7

5

ACCTTGGACT ACCCTGCCCG CGCCCATACT TTTGATGATT TCTGCCCAGA GTGCCGCCCC 60

CTTGGCCTTC AGGGCTGCGC TTTCCAGTCT ACTGTCGCTG AGCTTCAGCG CCTTAAGATG 120

AAGGTGGGTA AAACTCGGGA GTTGTAG 147

147 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

406.3-2, MEXICO, FIGURE 7

6

ACTTTTGATT ATCCGGGGCG GGCGCACACA TTTGATGACT TCTGCCCTGA ATGCCGCGCT 60

TTAGGCCTCC AGGGTTGTGC TTTCCAGTCA ACTGTCGCTG AGCTCCAGCG CCTTAAAGTT 120

AAGGTGGGTA AAACTCGGGA GTTGTAG 147

984 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

SG3, BURMA, FIGURE 7

7

GGTGCGGACG GGACTGCCGA GCTCACCACC ACGGCTGCTA CCCGCTTTAT GAAGGACCTC 60

TATTTTACTA GTACTAATGG TGTCGGTGAG ATCGGCCGCG GGATAGCCCT CACCCTGTTC 120

AACCTTGCTG ACACTCTGCT TGGCGGCCTG CCGACAGAAT TGATTTCGTC GGCTGGTGGC 180

CAGCTGTTCT ACTCCCGTCC CGTTGTCTCA GCCAATGGCG AGCCGACTGT TAAGTTGTAT 240

ACATCTGTAG AGAATGCTCA GCAGGATAAG GGTATTGCAA TCCCGCATGA CATTGACCTC 300

GGAGAATCTC GTGTGGTTAT TCAGGATTAT GATAACCAAC ATGAACAAGA TCGGCCGACG 360

CCTTCTCCAG CCCCATCGCG CCCTTTCTCT GTCCTTCGAG CTAATGATGT GCTTTGGCTC 420

TCTCTCACCG CTGCCGAGTA TGACCAGTCC ACTTATGGCT CTTCGACTGG CCCAGTTTAT 480

GTTTCTGACT CTGTGACCTT GGTTAATGTT GCGACCGGCG CGCAGGCCGT TGCCCGGTCG 540

CTCGATTGGA CCAAGGTCAC ACTTGACGGT CGCCCCCTCT CCACCATCCA GCAGTACTCG 600

AAGACCTTCT TTGTCCTGCC GCTCCGCGGT AAGCTCTCTT TCTGGGAGGC AGGCACAACT 660

AAAGCCGGGT ACCCTTATAA TTATAACACC ACTGCTAGCG ACCAACTGCT TGTCGAGAAT 720

GCCGCCGGGC ACCGGGTCGC TATTTCCACT TACACCACTA GCCTGGGTGC TGGTCCCGTC 780

TCCATTTCTG CGGTTGCCGT TTTAGCCCCC CACTCTGCGC TAGCATTGCT TGAGGATACC 840

TTGGACTACC CTGCCCGCGC CCATACTTTT GATGATTTCT GCCCAGAGTG CCGCCCCCTT 900

GGCCTTCAGG GCTGCGCTTT CCAGTCTACT GTCGCTGAGC TTCAGCGCCT TAAGATGAAG 960

GTGGGTAAAA CTCGGGAGTT GTAG 984

981 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

SG3, MEXICO, FIGURE 7

8

GCCGACGGGA CTGCGGAGCT GACCACAACT GCAGCCACCA GGTTCATGAA AGATCTCCAC 60

TTTACCGGCC TTAATGGGGT AGGTGAAGTC GGCCGCGGGA TAGCTCTAAC ATTACTTAAC 120

CTTGCTGACA CGCTCCTCGG CGGGCTCCCG ACAGAATTAA TTTCGTCGGC TGGCGGGCAA 180

CTGTTTTATT CCCGCCCGGT TGTCTCAGCC AATGGCGAGC CAACCGTGAA GCTCTATACA 240

TCAGTGGAGA ATGCTCAGCA GGATAAGGGT GTTGCTATCC CCCACGATAT CGATCTTGGT 300

GATTCGCGTG TGGTCATTCA GGATTATGAC AACCAGCATG AGCAGGATCG GCCCACCCCG 360

TCGCCTGCGC CATCTCGGCC TTTTTCTGTT CTCCGAGCAA ATGATGTACT TTGGCTGTCC 420

CTCACTGCAG CCGAGTATGA CCAGTCCACT TACGGGTCGT CAACTGGCCC GGTTTATATC 480

TCGGACAGCG TGACTTTGGT GAATGTTGCG ACTGGCGCGC AGGCCGTAGC CCGATCGCTT 540

GACTGGTCCA AAGTCACCCT CGACGGGCGG CCCCTCCCGA CTGTTGAGCA ATATTCCAAG 600

ACATTCTTTG TGCTCCCCCT TCGTGGCAAG CTCTCCTTTT GGGAGGCCGG CACAACAAAA 660

GCAGGTTATC CTTATAATTA TAATACTACT GCTAGTGACC AGATTCTGAT TGAAAATGCT 720

GCCGGCCATC GGGTCGCCAT TTCAACCTAT ACCACCAGGC TTGGGGCCGG TCCGGTCGCC 780

ATTTCTGCGG CCGCGGTTTT GGCTCCACGC TCCGCCCTGG CTCTGCTGGA GGATACTTTT 840

GATTATCCGG GGCGGGCGCA CACATTTGAT GACTTCTGCC CTGAATGCCG CGCTTTAGGC 900

CTCCAGGGTT GTGCTTTCCA GTCAACTGTC GCTGAGCTCC AGCGCCTTAA AGTTAAGGTG 960

GGTAAAACTC GGGAGTTGTA G 981

1311 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

C2, BURMA, FIGURE 7

9

AATTCAATAA CCTCGACGGA TGTTCGTATT TTAGTCCAGC CCGGCATAGC CTCTGAGCTT 60

GTGATCCCAA GTGAGCGCCT ACACTATCGT AACCAAGGCT GGCGCTCCGT CGAGACCTCT 120

GGGGTGGCTG AGGAGGAGGC TACCTCTGGT CTTGTTATGC TTTGCATACA TGGCTCACTC 180

GTAAATTCCT ATACTAATAC ACCCTATACC GGTGCCCTCG GGCTGTTGGA CTTTGCCCTT 240

GAGCTTGAGT TTCGCAACCT TACCCCCGGT AACACCAATA CGCGGGTCTC CCGTTATTCC 300

AGCACTGCTC GCCACCGCCT TCGTCGCGGT GCGGACGGGA CTGCCGAGCT CACCACCACG 360

GCTGCTACCC GCTTTATGAA GGACCTCTAT TTTACTAGTA CTAATGGTGT CGGTGAGATC 420

GGCCGCGGGA TAGCCCTCAC CCTGTTCAAC CTTGCTGACA CTCTGCTTGG CGGCCTGCCG 480

ACAGAATTGA TTTCGTCGGC TGGTGGCCAG CTGTTCTACT CCCGTCCCGT TGTCTCAGCC 540

AATGGCGAGC CGACTGTTAA GTTGTATACA TCTGTAGAGA ATGCTCAGCA GGATAAGGGT 600

ATTGCAATCC CGCATGACAT TGACCTCGGA GAATCTCGTG TGGTTATTCA GGATTATGAT 660

AACCAACATG AACAAGATCG GCCGACGCCT TCTCCAGCCC CATCGCGCCC TTTCTCTGTC 720

CTTCGAGCTA ATGATGTGCT TTGGCTCTCT CTCACCGCTG CCGAGTATGA CCAGTCCACT 780

TATGGCTCTT CGACTGGCCC AGTTTATGTT TCTGACTCTG TGACCTTGGT TAATGTTGCG 840

ACCGGCGCGC AGGCCGTTGC CCGGTCGCTC GATTGGACCA AGGTCACACT TGACGGTCGC 900

CCCCTCTCCA CCATCCAGCA GTACTCGAAG ACCTTCTTTG TCCTGCCGCT CCGCGGTAAG 960

CTCTCTTTCT GGGAGGCAGG CACAACTAAA GCCGGGTACC CTTATAATTA TAACACCACT 1020

GCTAGCGACC AACTGCTTGT CGAGAATGCC GCCGGGCACC GGGTCGCTAT TTCCACTTAC 1080

ACCACTAGCC TGGGTGCTGG TCCCGTCTCC ATTTCTGCGG TTGCCGTTTT AGCCCCCCAC 1140

TCTGCGCTAG CATTGCTTGA GGATACCTTG GACTACCCTG CCCGCGCCCA TACTTTTGAT 1200

GATTTCTGCC CAGAGTGCCG CCCCCTTGGC CTTCAGGGCT GCGCTTTCCA GTCTACTGTC 1260

GCTGAGCTTC AGCGCCTTAA GATGAAGGTG GGTAAAACTC GGGAGTTGTA G 1311

1308 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

C2, MEXICO, FIGURE 7

10

AATTCCATTA CTTCCACTGA TGTCAGGATT CTTGTTCAAC CTGGCATAGC ATCTGAATTG 60

GTCATCCCAA GCGAGCGCCT TCACTACCGC AATCAAGGTT GGCGCTCGGT TGAGACATCT 120

GGTGTTGCTG AGGAGGAAGC CACCTCCGGT CTTGTCATGT TATGCATACA TGGCTCTCCA 180

GTTAACTCCT ATACCAATAC CCCTTATACC GGTGCCCTTG GCTTACTGGA CTTTGCCTTA 240

GAGCTTGAGT TTCGCAATCT CACCACCTGT AACACCAATA CACGTGTGTC CCGTTACTCC 300

AGCACTGCTC GTCACTCCGC CCGAGGGGCC GACGGGACTG CGGAGCTGAC CACAACTGCA 360

GCCACCAGGT TCATGAAAGA TCTCCACTTT ACCGGCCTTA ATGGGGTAGG TGAAGTCGGC 420

CGCGGGATAG CTCTAACATT ACTTAACCTT GCTGACACGC TCCTCGGCGG GCTCCCGACA 480

GAATTAATTT CGTCGGCTGG CGGGCAACTG TTTTATTCCC GCCCGGTTGT CTCAGCCAAT 540

GGCGAGCCAA CCGTGAAGCT CTATACATCA GTGGAGAATG CTCAGCAGGA TAAGGGTGTT 600

GCTATCCCCC ACGATATCGA TCTTGGTGAT TCGCGTGTGG TCATTCAGGA TTATGACAAC 660

CAGCATGAGC AGGATCGGCC CACCCCGTCG CCTGCGCCAT CTCGGCCTTT TTCTGTTCTC 720

CGAGCAAATG ATGTACTTTG GCTGTCCCTC ACTGCAGCCG AGTATGACCA GTCCACTTAC 780

GGGTCGTCAA CTGGCCCGGT TTATATCTCG GACAGCGTGA CTTTGGTGAA TGTTGCGACT 840

GGCGCGCAGG CCGTAGCCCG ATCGCTTGAC TGGTCCAAAG TCACCCTCGA CGGGCGGCCC 900

CTCCCGACTG TTGAGCAATA TTCCAAGACA TTCTTTGTGC TCCCCCTTCG TGGCAAGCTC 960

TCCTTTTGGG AGGCCGGCAC AACAAAAGCA GGTTATCCTT ATAATTATAA TACTACTGCT 1020

AGTGACCAGA TTCTGATTGA AAATGCTGCC GGCCATCGGG TCGCCATTTC AACCTATACC 1080

ACCAGGCTTG GGGCCGGTCC GGTCGCCATT TCTGCGGCCG CGGTTTTGGC TCCACGCTCC 1140

GCCCTGGCTC TGCTGGAGGA TACTTTTGAT TATCCGGGGC GGGCGCACAC ATTTGATGAC 1200

TTCTGCCCTG AATGCCGCGC TTTAGGCCTC CAGGGTTGTG CTTTCCAGTC AACTGTCGCT 1260

GAGCTCCAGC GCCTTAAAGT TAAGGTGGGT AAAACTCGGG AGTTGTAG 1308

102 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

406.4-2, BURMA, FIGURE 7

11

GCCAACCCGC CCGACCACTC GGCTCCGCTT GGCGTGACCA GGCCCAGCGC CCCGCCGTTG 60

CCTCACGTCG TAGACCTACC ACAGCTGGGG CCGCGCCGCT AA 102

102 base pairs

nucleic acid

double

linear

cDNA to mRNA

NO

406.4-2, MEXICO, FIGURE 7

12

GCCAACCAGC CCGGCCACTT GGCTCCACTT GGCGAGATCA GGCCCAGCGC CCCTCCGCTG 60

CCTCCCGTCG CCGACCTGCC ACAGCCGGGG CTGCGGCGCT GA 102

48 amino acids

amino acid

linear

protein

NO

406.3-2, BURMA, FIGURE 9

13

Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro

1 5 10 15

Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val

20 25 30

Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu

35 40 45

48 amino acids

amino acid

linear

protein

NO

406.3-2, MEXICO, FIGURE 9

14

Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp Phe Cys Pro

1 5 10 15

Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val

20 25 30

Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr Arg Glu Leu

35 40 45

327 amino acids

amino acid

linear

protein

NO

SG3, BURMA, FIGURE 9

15

Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe

1 5 10 15

Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly

20 25 30

Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly

35 40 45

Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr

50 55 60

Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr

65 70 75 80

Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His

85 90 95

Asp Ile Asp Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn

100 105 110

Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro

115 120 125

Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala

130 135 140

Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr

145 150 155 160

Val Ser Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala

165 170 175

Val Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro

180 185 190

Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu

195 200 205

Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr

210 215 220

Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn

225 230 235 240

Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly

245 250 255

Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser

260 265 270

Ala Leu Ala Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His

275 280 285

Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly

290 295 300

Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys

305 310 315 320

Val Gly Lys Thr Arg Glu Leu

325

327 amino acids

amino acid

linear

protein

NO

SG3, MEXICO, FIGURE 9

16

Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe

1 5 10 15

Met Lys Asp Leu His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly

20 25 30

Arg Gly Ile Ala Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly

35 40 45

Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr

50 55 60

Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr

65 70 75 80

Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His

85 90 95

Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn

100 105 110

Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro

115 120 125

Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala

130 135 140

Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr

145 150 155 160

Ile Ser Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala

165 170 175

Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro

180 185 190

Leu Pro Thr Val Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu

195 200 205

Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr

210 215 220

Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn

225 230 235 240

Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly

245 250 255

Ala Gly Pro Val Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser

260 265 270

Ala Leu Ala Leu Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His

275 280 285

Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly

290 295 300

Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys

305 310 315 320

Val Gly Lys Thr Arg Glu Leu

325

436 amino acids

amino acid

linear

protein

NO

C2, BURMA, FIGURE 9

17

Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile

1 5 10 15

Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln

20 25 30

Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr

35 40 45

Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Leu Val Asn Ser Tyr

50 55 60

Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu

65 70 75 80

Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val

85 90 95

Ser Arg Tyr Ser Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp

100 105 110

Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp

115 120 125

Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly Arg Gly Ile

130 135 140

Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro

145 150 155 160

Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro

165 170 175

Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val

180 185 190

Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp

195 200 205

Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu

210 215 220

Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val

225 230 235 240

Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr

245 250 255

Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser Asp

260 265 270

Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg

275 280 285

Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser Thr

290 295 300

Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys

305 310 315 320

Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn

325 330 335

Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala Gly

340 345 350

His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro

355 360 365

Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser Ala Leu Ala

370 375 380

Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp

385 390 395 400

Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe

405 410 415

Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys

420 425 430

Thr Arg Glu Leu

435

435 amino acids

amino acid

linear

protein

NO

C2, MEXICO, FIGURE 9

18

Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile

1 5 10 15

Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln

20 25 30

Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr

35 40 45

Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr

50 55 60

Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu

65 70 75 80

Glu Leu Glu Phe Arg Asn Leu Thr Thr Cys Asn Thr Asn Thr Arg Val

85 90 95

Ser Arg Tyr Ser Ser Thr Ala Arg His Ser Ala Arg Gly Ala Asp Gly

100 105 110

Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu

115 120 125

His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala

130 135 140

Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr

145 150 155 160

Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val

165 170 175

Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu

180 185 190

Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His Asp Ile Asp Leu

195 200 205

Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln

210 215 220

Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu

225 230 235 240

Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp

245 250 255

Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Ile Ser Asp Ser

260 265 270

Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser

275 280 285

Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Pro Thr Val

290 295 300

Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys Leu

305 310 315 320

Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr

325 330 335

Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His

340 345 350

Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly Ala Gly Pro Val

355 360 365

Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser Ala Leu Ala Leu

370 375 380

Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp

385 390 395 400

Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln

405 410 415

Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr

420 425 430

Arg Glu Leu

435

660 amino acids

amino acid

linear

protein

NO

ORF 2, BURMA, FIGURE 9

19

Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro Met

1 5 10 15

Leu Pro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg

20 25 30

Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser

35 40 45

Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Pro

50 55 60

Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg Val Arg Gln Pro Ala

65 70 75 80

Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln Ala Gln Arg Pro Ala Val

85 90 95

Ala Ser Arg Arg Arg Pro Thr Thr Ala Gly Ala Ala Pro Leu Thr Ala

100 105 110

Val Ala Pro Ala His Asp Thr Pro Pro Val Pro Asp Val Asp Ser Arg

115 120 125

Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr

130 135 140

Ser Ser Val Ala Thr Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu

145 150 155 160

Ser Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala

165 170 175

Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile

180 185 190

Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser

195 200 205

Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met

210 215 220

Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile

225 230 235 240

Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln

245 250 255

Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr

260 265 270

Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Leu Val Asn Ser Tyr

275 280 285

Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu

290 295 300

Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val

305 310 315 320

Ser Arg Tyr Ser Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp

325 330 335

Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp

340 345 350

Leu Tyr Phe Thr Ser Thr Asn Gly Val Gly Glu Ile Gly Arg Gly Ile

355 360 365

Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro

370 375 380

Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro

385 390 395 400

Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val

405 410 415

Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp

420 425 430

Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu

435 440 445

Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val

450 455 460

Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr

465 470 475 480

Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser Asp

485 490 495

Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg

500 505 510

Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser Thr

515 520 525

Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys

530 535 540

Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn

545 550 555 560

Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala Gly

565 570 575

His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro

580 585 590

Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser Ala Leu Ala

595 600 605

Leu Leu Glu Asp Thr Leu Asp Tyr Pro Ala Arg Ala His Thr Phe Asp

610 615 620

Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe

625 630 635 640

Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys

645 650 655

Thr Arg Glu Leu

660

659 amino acids

amino acid

linear

protein

NO

ORF 2, MEXICO, FIGURE 9

20

Met Arg Pro Arg Pro Leu Leu Leu Leu Phe Leu Leu Phe Leu Pro Met

1 5 10 15

Leu Pro Ala Pro Pro Thr Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg

20 25 30

Arg Ser Gly Gly Thr Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser

35 40 45

Gln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Pro

50 55 60

Asp Val Ala Ala Ala Ser Gly Ser Gly Pro Arg Leu Arg Gln Pro Ala

65 70 75 80

Arg Pro Leu Gly Ser Thr Trp Arg Asp Gln Ala Gln Arg Pro Ser Ala

85 90 95

Ala Ser Arg Arg Arg Pro Ala Thr Ala Gly Ala Ala Ala Leu Thr Ala

100 105 110

Val Ala Pro Ala His Asp Thr Ser Pro Val Pro Asp Val Asp Ser Arg

115 120 125

Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr

130 135 140

Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu

145 150 155 160

Asn Pro Pro Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala

165 170 175

Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile

180 185 190

Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser

195 200 205

Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met

210 215 220

Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile

225 230 235 240

Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln

245 250 255

Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu Ala Thr

260 265 270

Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr

275 280 285

Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu

290 295 300

Glu Leu Glu Phe Arg Asn Leu Thr Thr Cys Asn Thr Asn Thr Arg Val

305 310 315 320

Ser Arg Tyr Ser Ser Thr Ala Arg His Ser Ala Arg Gly Ala Asp Gly

325 330 335

Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu

340 345 350

His Phe Thr Gly Leu Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala

355 360 365

Leu Thr Leu Leu Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr

370 375 380

Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val

385 390 395 400

Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu

405 410 415

Asn Ala Gln Gln Asp Lys Gly Val Ala Ile Pro His Asp Ile Asp Leu

420 425 430

Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln

435 440 445

Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu

450 455 460

Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp

465 470 475 480

Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Ile Ser Asp Ser

485 490 495

Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser

500 505 510

Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Pro Thr Val

515 520 525

Glu Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly Lys Leu

530 535 540

Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr

545 550 555 560

Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His

565 570 575

Arg Val Ala Ile Ser Thr Tyr Thr Thr Arg Leu Gly Ala Gly Pro Val

580 585 590

Ala Ile Ser Ala Ala Ala Val Leu Ala Pro Arg Ser Ala Leu Ala Leu

595 600 605

Leu Glu Asp Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp

610 615 620

Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln

625 630 635 640

Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr

645 650 655

Arg Glu Leu

33 amino acids

amino acid

linear

protein

NO

406.4-2, BURMA, FIGURE 8

21

Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro Ser

1 5 10 15

Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly Pro Arg

20 25 30

Arg

33 amino acids

amino acid

linear

protein

NO

406.4-2, MEXICO, FIGURE 8

22

Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gly Glu Ile Arg Pro Ser

1 5 10 15

Ala Pro Pro Leu Pro Pro Val Ala Asp Leu Pro Gln Pro Gly Leu Arg

20 25 30

Arg